The finding that behavioral method implementing a short sequence of stress arousals and its resolutions can rectify persistent behavioral changes in multiple models of depression highlights the feasibility of fighting adaptively altered stress gains with behavioral stress. Chronic restraint produces depressive behaviors that last for more than 3 months38,39. Despite those adaptive changes, RS5, but not RS10 or RS15, rectified depressive behaviors as did the antidepressant imipramine (Fig. 1). RS5 treatment did not produce anti-depressive effects when the HPA axis was blocked (Fig. 3 and Extended Data Fig. 3). Repeated injection with low-dose GC (0.1 mg/kg) recapitulated the effects of RS5 (Fig. 2h–l). Paradoxically, however, repeated injection with GC at a dose higher than that induced by 10-min or 15-min restraint, and repeated injection with even 1.0 mg/kg of GC, which was comparable to the GC level induced by 2-h restraint (Fig. 2a,g), also produced anti-depressive effects (Fig. 2h–l). These results suggest that behavioral appraisal by implementing a short sequence of stress produces anti-depressive effects via a GC-dependent mechanism. However behavioral method has a limited window to produce anti-depressive effects.
Chronic stress changes various brain regions beyond the homeostatic range by activating the HPA axis16,18,19. Patients with depression have increased basal serum GC levels40,41. In contrast, RU486 (mifepristone), a GR antagonist, is beneficial for patients with psychotic depression42 and bipolar disorder14. Mice exposed to chronic stress have increased basal serum GC levels (Fig. 2). Administration of high-dose GC in drinking water (35 μg/ml/day) for 4 weeks43 or subcutaneous injection of GC at a dose of 10, 20 or 40 mg/kg/day for 21 days44 in rats mirrors the stress-induced dysfunction of the HPA axis and produces depressive-like behaviors. Therefore, GC is regarded as a mediator of chronic stress15,16,17.
In the present study, we demonstrated that repeated treatment with a short sequence of behavioral stress or repeated injection with GC (0.1 – 1.0 mg/kg/day) produced anti-depressive effects (Figs. 1 and 2h–l) and reversed stress-induced molecular changes (Fig. 5) as did imipramine. The key findings are summarized in Fig. 10. These results raise the following important and related points. First, the fact that GC induction by behavioral stress and exogenous GC resolved existing stress gains suggests that GC functions as a stress modifier, which contradicts the classical conception that GC is a stress mediator. Although when and how GC functions as a stress modifier need to be studied in more detail, we speculate that repeated weak stress can restore stress coping ability, presumably by repeatedly boosting the feedback and feedforward regulatory mechanisms of the HPA axis (Fig. 10b,c,e). This possibility does not conflict with the classical conception that chronically imposed GC produces cumulative effects on stress gains due to the points described below. It will be worth studying whether the circadian oscillation of basal GC levels45,46, which is disrupted in patients with depression40,41, functions as a stress modifier by performing a daily reset of the stress coping system. In healthy individuals, the basal serum GC levels vary throughout the day, with the highest in the early morning and then falling throughout the day to the late evening. Second, repeated treatment with the behavioral stress or repeated treatment with GC could be used as an antidepressant strategy. Although the behavioral method has a narrow window to afford therapeutic effects, it could have an advantage over the pharmacological method. On the other hand, challenging with GC provides a more wider and more versatile therapeutic window to resolve existing stress gains. The profound therapeutic effects of behavioral stress and exogenous GC demonstrated in this study warrant further investigation. Third, the finding that repeated weak stress or treatment with even high-dose GC did not strengthen existing stress gains but resolved them (Figs. 1 and 2), raises the possibility that prior stress gains might become transiently deconsolidated and labile upon new stress inputs or GC flux. As stress-induced adaptive changes are deconsolidated by GC, brain cells appear to restore their normal homeostatic stability and physiological function to produce normal behavioral outputs. It will be worth studying the key factors and underlying signaling networks that regulate GC-dependent changes and the mechanisms of homeostatic restoration.
Chronic stress increased the p-CaMKII level, which downregulated GR expression in PL neurons (Fig. 6a,p). Local inhibition of CaMKIIα in the PL produced anti-depressive effects in CRST-treated mice (Fig. 6l–s), and siRNA-mediated inhibition of GR in the PL of normal mice increased Fkbp5 and produced depressive-like behaviors (Fig. 7o–t). p-CaMKIIα upregulation in PL neurons appears to be caused by stress-induced downregulation of GABAA receptors (Fig. 7). Fkbp5, in coordination with Hsp90, negatively regulates GR nuclear translocation33,47, so a stress-induced increase of p-CaMKII in the PL facilitates a vicious cycle in the GR↓ à Fkbp5↑ pathway. These results suggest that stress-induced upregulation of p-CaMKIIα in the PL is a critical player mediating the effects of chronic stress on depressive behaviors.
Our results indicate that PL neurons and their associated neural systems, including the NAc, BLA, and BNST, compose the critical neuronal nodes and edges that support stress-induced depression and the modification of stress-induced changes by strategic weak stress, as summarized (Fig. 10b-e). Glutamate/glutamine levels and the neural activity of glutamatergic neurons in the mPFC are reduced in CUMS, CRST, and CSDS-induced depression models in mice48,49,50. Chemogenetic activation of PL neurons facilitated anti-depressive effects and suppressed the stress-induced increase in basal GC levels, whereas chemogenetic inhibition of PL neurons during RS5 dissipated its anti-depressive effects (Figs. 8 and Extended Data Fig. 7). Furthermore, chemogenetic inhibition of the PLà NAc, PLà BLA or PLà dBNST circuits during RS5 blocked its anti-depressive effects (Fig. 9), suggesting that activation of those circuits is required for RS5 to produce anti-depressive effects. Previous studies reported that activation of the PL51, the PLà NAc circuit52 or the PLà BLA circuit53 produced anti-depressive effects, which is partly consistent with our results. Interestingly, however, the PLà dBNST circuit played a role in the recovery of basal GC levels by RS5, whereas the PLà NAc and PLà BLA circuits did not (Fig. 9), raising the question if the latter cases, which did not recover normal HPA function, produce stable recovery from stress-induced changes. Overall, our results suggest that although the PL is the key area recruited by RS5, the PLà NAc, PLà BLA, and PLàdBNST pathways comprise the critical neural nodes and circuits that support the behavioral effects of RS5, and the PLàdBNST circuit supports the recovery of the HPA axis by RS5 (Fig. 10b-e).
Stress inoculation is a pretreatment strategy that improves subsequent stress coping and emotional regulation45. Stress inoculation in mice, by placing them behind a mesh-screen barrier in a cage containing an aggressor mouse for 15 min, enhances subsequent stress coping behavior and cognitive function55. Another form of stress inoculation, called predictable chronic mild stress (PCMS), uses 5-min restraint for 28 days to improve mood, hippocampal neurogenesis and memory in rats56. PCMS treatment of rats in their early adolescence increases resilience to chronic unpredictable mild stress in adulthood57. In those studies, stress inoculation or PCMS is regarded as an immunization to enhance coping ability for future stress54. PCMS treated for 28 days induced anti-depressive effects in rats56. In contrast, our RS5 treatment (daily 5-min restraint for 14 days) in normal mice did not induce depressive behaviors (Fig. 1a-g), Therefore, it will be worth studying that a pretreatment paradigm of daily 5-min restraint for 7-14 days in normal mice would also produce resiliency to future chronic stress. It is possible that stress inoculation/PCMS and RS5 could commonly have a certain neural mechanism. Nonetheless, our experimental procedure provoking weak stress deals with a therapeutic strategy, whereas stress inoculation/PCMS is preventative.
Fig. 1
Fig. 1 Repeated treatment with a short sequence of behavioral stress produces anti-depressive effects in stress-induced models of depression.
a, Experimental design. Mice were treated with 5-min or 2-h restraint for 14 days. CRST mice were treated with 5-min, 10-min, or 15-min restraint or imipramine for 14 days (RS5, RS10, RS15, and IMI, respectively), and then given behavioral tests.
b–g, Representative tracks and % time spent in the target and non-target chambers in the SIT (b), sucrose preference in the SPT (c), and immobility time in the TST (d) and FST (e) for the indicated groups on post-stress days 15-17. K-Means clustering (k=2) of individuals in the SIT x SPT x [TST x FST] matrix (f) and % composition of each group in the clusters (g). PCA was used for dimension reduction of the TST x FST components (PVE, 81.3%) (n = 9-10 per group).
h–I, Immobility time in the TST (h) and FST (i) for the indicated groups on post-stress days 43 and 44 (n = 8-10 per group). j, Experimental design. The susceptible mice were treated with RS5, and then given behavioral tests.
k–o, Mice susceptible or resilient to CSDS were separated by the sociability ratio (k). % time spent in the target chamber in the two-chamber SIT (l), sucrose preference in the SPT (m), and immobility time in the TST (n) and FST (o) for the indicated groups (n = 8-11 per group).
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05; **, ##, p < 0.01 (One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Fig. 2
Fig. 2 RS5 and low-dose CORT normalize stress-induced dysregulation of the HPA axis.
a, Serum CORT levels in mice treated with 5-min, 15-min, 60-min, and 120-min restraint (n = 8-9 per group).
b–f, Experimental design (b). Time course of serum CORT levels in the indicated groups after exposure to a single 5-min restraint (S5) (Exp #1) (c), and the area under the curve (AUC) between -5 min and 120 min (d) (n = 7-8 per group). Basal serum CORT levels (e) and CRH and AVP expression levels in the PVN (f) of the indicated groups (Exp #2) (n = 7-8 per group).
g, Changes in serum CORT levels in mice after CORT injection (n = 8-9 per group).
h–p, Experimental design (h). CORT was injected for 14 days (0.1, 0.5, 1.0 mg/kg/day, i.p.) in CRST mice. Behavioral performance in the SIT (i), SPT (j), TST (k), and FST (l) for the indicated groups (n = 10 per group). Basal serum CORT levels (n = 8 per group) (m) and adrenal gland (AG) weight (n = 10 per group) (n) of the indicated groups on post-stress day 22. K-Means clustering of individuals in the [AG weight] x [SIT x SPT] x [TST x FST] matrix (o) and % composition of each group in the clusters (p). PCA was used for dimensional reduction of the SIT and SPT components (PVE; 76.0%) and the TST and FST components (PVE; 73.4%).
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05; **, ##, p < 0.01 (One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Fig. 3
Fig. 3 Adrenalectomy (ADX) blocks the antidepressive effects of RS5 in CRST mice.
a, Experimental design for treatment with CRST, ADX or sham surgery, followed by RS5 treatment and behavioral tests. Red arrows (↓) in Exp #1, Serum collection point. Mice were sacrificed after exposure to a single 5-min restraint (S5).
b–h, The basal CORT levels at -5 min, and the time course of serum CORT levels in the indicated groups after exposure to S5 (Exp #1) (b) (n = 7-10 per group). Social interaction in the SIT (c), sucrose preference in the SPT (d), and immobility time in the TST (e) and FST (f) for the indicated groups (Exp #2). K-Means clustering of individuals in the SIT x SPT x [TST x FST] matrix (g) and % composition of each group in the clusters (h). The TST and FST components were transformed into linear eigenvectors using PCA (PVE; 78.2%) (n = 7-10 per group).
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST; +, difference compared to CRST+RS5. *, #, +, p < 0.05; **, ##, ++, p < 0.01 (One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Fig. 4
Fig. 4 Repeated treatment with a short sequence of behavioral stress recruits the brain regions regulating stress coping in CRST mice.
a, Experimental design. CRST mice were treated with a single 5-min or 15-min restraint (S5 and S15, respectively) on post-stress day 8, and then sacrificed, 20 min and 10 min later, respectively. Another group of CRST mice was treated with 5-min or 15-min restraint for 8 days (S5x8d and S15x8d, respectively), and then sacrificed, 20 min and 10 min, respectively, after the last restraint.
b,c, Quantification of c-Fos expression levels in the PL, BLA, NAcc, vSub, dBNST, ventral BNST (vBNST), and PVN (b). Photomicrographs showing c-Fos expression in the PL (red box) (C) of the S5, S15, S5x8d, and S15x8d groups (n = 4-6 per group).
d–g, Photomicrographs showing c-Fos induction in GLU-4-positive or GAD67-positive neurons in the PL (d), and their quantification levels (e–g) (n = 6 animals for each).
The details are shown in Supplementary Table 1. Data are mean ± SEM. *, **, difference compared to control; #, difference compared to CRST; +, difference between indicated groups. **, ##, ++, p < 0.01 (one-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Fig. 5
Fig. 5 RS5 treatment reverses stress-induced changes in gene expression profiles in the PL.
a–c, Experimental design (a). Heatmap showing the expression profiles were changed by ≥1.2-fold after CRST (b) and reversed by RS5 treatment in CRST mice (c), and follow-up unbiased alignment with the expression levels of the respective genes changed by RS5 (b) and CRST (c). ↑ and ↓, indicate up- and down-regulation, respectively.
d–f, GO enrichment analysis with weighted K-Means clustering identified the clusters which carry the functional modules labeled with the GO terms “response to stress” or “response to glucocorticoid” (d, e). The PPI networks formed by the members of clusters 5 (orange, 28 genes) and 7 (green, 24 genes) and 16 genes (black) from an extended search of the STRING database at a confidence level of >0.700 (f).
g,h, Expression levels of Nr3c1, Nr3c2, Fkbp5, Fkbp4, Hsp90aa1, and Hsp90ab1 (g), and Dusp1, CaMKIIα, Mapk3, and Mapk1 (h) in the PL of the indicated groups (n = 8-12 per group).
i–n, Immunofluorescence images showing GR (red) and Fkbp5 (green) expression in the PL from the indicated groups (i). DAPI, blue. Quantification levels of total GR/DAPI (j), Fkbp5/DAPI (k), and DAPI intensity (l) in individual cells (n = 4-6 animals per group; n = 248 – 309 per group). Differential expression of nuclear GR and Fkbp5 in individual cells (m). Two distinct clusters with centroids classified by K-Means clustering, and the regression values (m) and % composition of individuals cells of each group (n) in the clusters were indicated.
o–t, Experimental design (o). siRNA-mediated knockdown of GR in the PL, and subsequent behavioral tests. Red arrow, time point for tissue prep. Transcript levels of GR (p) and Fkbp5 (q) in the PL and of CRH and AVP (r) in the PVN. Immobility time in the TST (s) and FST (t) for each group.
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05; **, ##, p < 0.01 (One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Fig. 6
Fig. 6 RS5 restores increased p-CaMKIIα expression in PL neurons.
a–c, Western blots showing expression levels of p-CaMKIIα, CaMKIIα, p-ERK1/2, ERK1/2, and β-actin in the PL of the indicated groups (a). Quantification levels of p-CaMKIIα (b) and p-ERK1/2 expression (c) (n=6 animals per group, 4 repeats).
d,e, Immunofluorescence staining of p-CaMKIIα (green) in PL neurons stained with GLU-4 or GAD67 (red) (d). Quantification levels (e) (n = 4 per group).
f–k, Immunofluorescence staining of p-CaMKIIα (red) and total GR (green) in PL neurons (f). DAPI, blue. Differential expression of p-CaMKIIα and total GR in individual cells of the indicated groups (g). Two distinct clusters with centroids and regression values (g) and the % composition of individuals cells of each group in the clusters (h). Quantification levels of p-CaMKIIα (i), GR (j) and DAPI (k) intensity in individual cells (n = 4-6 animals per group; n = 345 – 390 per group).
l–s, Experimental design (l). siRNA-mediated knockdown of CaMKIIα (m), ERK1 (n), and ERK2 (o) in the PL of CRST-treated mice. Red arrow, time point for tissue prep. Transcript levels of GR and Fkbp5 (p) in the PL of the indicated groups. Behavioral performance in the SIT (q), TST (r), and FST (s) for the indicated groups.
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05, **, ##, p < 0.01 (Student’s t-test; One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Fig. 7
Fig. 7 CaMKIIα in the PL is a critical player regulating depressive behaviors.
a, Transcript levels of GABRα1 and GABRβ2 in the PL of the indicated groups. Sample groups were prepared from the experiments depicted in Fig. 4g,h (n = 7-12 per group).
b–g, Experimental design (b). The picrotoxin (PTX) was infused using a pre-implanted cannula into the PL of mice subjected to CRST and RS5. Behavior tests were carried out between 60 and 90 min after drug infusion on post-stress day 22 (b). Western blots showing the expression levels of p-CaMKIIα, CaMKIIα, p-ERK1/2, ERK1/2, and β-actin in the PL of the indicated groups (c). Quantification levels of p-CaMKIIα (d) and p-ERK1/2 (e) (n = 6 mice per group, 3 repeats). Behavioral performance in the TST (f) and FST (g) for the indicated groups (n = 8 per group).
h–m, Experimental design (h). siRNA-mediated knockdown of CaMKIIα (i) and ERK1 (j) in the PL. Behavioral tests were carried out 2 days later in the order SIT, TST, and FST. Red arrow, time point for tissue prep. Behavioral performance in the SIT (k), TST (l), and FST (m) for the indicated groups (n = 8 per group).
n,o, Immunofluorescence staining of p-ERK1/2 (green) expression in the PL stained with GLU-4 or GAD67 (red) (n). Quantification levels of colocalization (o) (n = 4 per group).
p–s, Experimental design (p). KN-62 (CaMKIIα inhibitor, 2.5 nmol/injection), or Veh was infused into the PL through a pre-implanted cannula. After 60 min, behavioral tests were carried out in the order SIT, TST and FST. Behavioral performance in the SIT (q), TST (r), and FST (s) for the indicated groups (n = 7 per group).
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05, **, ##, p < 0.01 (Student’s t-test; One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Fig. 8
Fig. 8 Chemogenetic inhibition of PL neurons blocks the anti-depressive effects of RS5.
a, Experimental design. Mice injected in the PL with AAV8-CaMKIIα-hM4D(Gi)-mCherry inhibitory vector were subjected to CRST, followed by RS5 treatment with Veh or CNO injection.
b–e, Injection of the AAV8 vector and resulting mCherry expression in the PL (b). S5-induced c-Fos expression levels in the PL after CNO treatment (Exp #1) (c and d) (n = 4-6 per group). Basal CORT levels in the indicated groups on post-stress day 22 (e) (n = 7-8 per group).
f–k, Behavioral performance in the SIT (f), SPT (g), TST (h), and FST (i) for the indicated groups (Exp #2). K-Means clustering of individuals in the SIT x SPT x [TST x FST] matrix (J) and % composition of each group in the clusters (K) (n = 7-9 per group).
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05, **, ##, p < 0.01 (Student’s t-test; one-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Fig. 9
Fig. 9 Chemogenetic inhibition of PL neurons projecting to the dBNST, BLA or NAcc blocks the anti-depressive effects of RS5.
a, Experimental design. Mice injected in the PL with the AAV8-hSyn-hM4D(Gi)-mCherry and retrograde Cre vector in the dBNST, BLA, or NAcc were subjected to CRST, followed by RS5 treatment with Veh or CNO injection.
b,c, Injection of the AAV8 vector and resulting mCherry expression in the PL (b) and the PLàdBNST circuit (c), which was labeled with hM4D(Gi) by injection of viral vectors (a).
d–f, S5-induced c-Fos expression levels in the dBNST (d and e) and PVN (f) after CNO treatment (Exp #1) (n = 4 per group). c-Fos, red; GAD67, green; DAPI, blue.
g, Basal CORT levels in the indicated groups on post-stress day 22 (n = 8 per group).
h–m, Behavioral performance in the SIT (h), SPT (i), TST (j), and FST (k) for the indicated groups (Exp #2). K-Means clustering of individuals in the SIT x SPT x [TST x FST] matrix (l) and % composition of each group in the clusters (m) (n = 8 per group).
n,o, The PLàBLA (n) or PLàNAcc (o) circuits, which were labeled with hM4D(Gi) by injection of viral vectors (a).
p–s, S5-induced c-Fos expression levels in the BLA (n = 3-4 per group) (p and q) and NAcc (n = 3-4 per group) (r and s) after CNO treatment (Exp #1).
t, Basal CORT levels in the indicated groups on post-stress day 22 (n = 10 per group).
u–x, Behavioral performance in the SIT (u), SPT (v), TST (w), and FST (x) for the indicated groups (Exp #2).
Data are mean ± SEM. Gray circles represent individual data points. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05; **, ##, p < 0.01 (Student’s t-test; One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Fig. 10
Fig. 10 A summary and hypothetical model for the recovery of adaptively changed stress gains by treatment with strategic behavioral stress or GC.
a, Chronic stress produces adaptive changes in the brain, and cumulative effects of chronic stress cause persistent depressive behavior. Repeated treatment with a short sequence of behavioral stress or GC in mice subjected to chronic stress reverses the stress-induced adaptive changes and rescues depressive behavior.
b, The schematic presentation of the brain with normal activity of PL outputs (PLàNAcc, PLàdBNST, and PLàBLA) and the limbic system, and normal activity of the HPA axis including basal GC release at a normal circadian cycle and its feedforward effects on the PL.
c,d, Chronic stress produces the PL overstimulated primarily due to increased GC (c), which results in genome-wide gene expression alteration (d). The reversal of the stress-induced changes by RS5 is included here. As a result, the neural activities of PL outputs (PLàNAcc, PLàdBNST, and PLàBLA) and the limbic system are downregulated or altered, and the basal GC release is enhanced and trailed off from a normal circadian cycle (c). The expression levels of GR, GABAR, NMDAR, p-CaMKIIa and Fkbp5 in the PL are changed by chronic stress, and their physiological effects on depressive behaviors are characterized in the present study (d).
e, Repeated treatment with behavioral stress or GC reverses the stress-induced altered neural activities of PL outputs, the altered gene expression profiles, the altered activity of the HPA axis including basal GC release, and the altered feedforward GC effects on the PL.
Extended Data Fig. 1
Extended Data Fig. 1 Dosing analysis for the repeatability of weak stress that produces anti-depressive effects.
a, Experimental design. CRST mice were treated with daily 5-min restraint for 3, 5, 7, or 14 days (S5x3d, S5x5d, S5x7d, and S5x14d, respectively) followed by behavioral tests.
b–f, Time spent in the target chamber in the SIT (b), and immobility time in the TST (c) and FST (d) for the indicated groups. K-Means clustering (k = 2) of individuals in the SIT x TST x FST matrix (e) and % composition of each group in the clusters (f) (n = 8-10 per group).
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05; **, ##, p < 0.01 (One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Extended Data Fig. 2
Extended Data Fig. 2 Repeated treatment with a short sequence of behavioral stress produces anti-depressive effects in ICR mice.
a,b, Experimental design (a). Pregnant ICR females were exposed to daily 2-h restraint from E8.5 until delivery (maternal stress or MS group), and their pups (MS pups) were grown to adulthood. Pregnant normal ICR females with no stress (normal or N group) and their pups (N pups) were prepared as controls. Beginning at 7 weeks of age, the MS pups and N pups were randomly allocated to receive CRST or CRST+RS5 for 14 days (b).
c–h, Time spent in the target chamber in the SIT (c), sucrose preference in the SPT (d), and immobility time in the TST (e) and FST (f) for the indicated groups, and their controls. PCA and K-Means cluster analysis of individual animals in the SIT x SPT x [TST x FST] matrix (g) and % composition in each cluster (h). CRST-treated N and MS groups were shifted from the cluster containing CRST mice to the cluster containing the control. The TST and FST components were transformed in a linear dimension by PCA (PVE; 75.6%) (n = 9-10 per group).
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05; **, ##, p < 0.01 (One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Extended Data Fig. 3
Extended Data Fig. 3 Pharmacological suppression of HPA axis activation blocks the antidepressive effects of RS5.
a, Experimental design. Mice were treated with CRST, followed by daily 5-min restraint 30 min after NBI27914 or RU486 injection for indicated days, and subsequent behavioral tests.
b–h, Basal serum CORT levels in the indicated groups (b) (n = 7-8 per group). Behavioral performance in the SIT (c), SPT (d), TST (e), and FST (f) for the indicated groups. K-Means clustering of individuals in the SIT x SPT x [TST x FST] matrix (g) and % composition of each group in the clusters (h). The TST and FST components were transformed into a one-dimensional variable using PCA (PVE; 74.4%) (n = 7-10 per group).
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST; +, difference compared to CRST+RS5; @, difference compared to the indicated group. *, #, +, @, p < 0.05; **, ##, ++, @@, p < 0.01; n.s., not significant (One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Extended Data Fig. 4
Extended Data Fig. 4 Low-dose CORT treatment activates the brain regions regulating stress coping in CRST mice.
a, Experimental design. Mice were subjected to CRST, followed by CORT injection (blue arrows) at 0.1, 0.5, or 1.0 mg/kg/day for 7 days and then sacrificed 25 min after an additional CORT injection on post-stress day 8. Red arrow, time point for tissue prep.
b, c-Fos expression levels induced by CORT injection in the PL, BLA, NAcc, vSub, dBNST, vBNST, and PVN (n = 4-6 per group).
c, Photomicrographs showing c-Fos expression in the PL, BLA, NAcc, vSub, dBNST, vBNST, and PVN (red boxes) of the CRST control, CRST+CORT (0.1 mg/kg), CRST+CORT (0.5 mg/kg), and CRST+CORT (1.0 mg/kg) groups.
The details are shown in Supplementary Table 1. Data are mean ± SEM. *, **, difference compared to control. *, p < 0.05; **, p < 0.01 (one-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Extended Data Fig. 5
Extended Data Fig. 5 Analysis of gene expression profiles and protein–protein interaction networks in the PL of mice exposed to CRST and RS5 treatment.
a, Serial K-Means clustering was used to group the 986 identified genes into featured clusters. Each increase in k value added a new cluster, and its members were mostly supplied from the largest cluster. The cognate inter-clusters are marked with the same color code. C, cluster. Concerning the classification with k = 8, cluster 1 (gray) contained 622 genes; cluster 2 (scarlet) contained 71 genes; cluster 3 (yellow) contained 39 genes; cluster 4 (light green) contained 29 genes; cluster 5 (orange) contained 28 genes; cluster 6 (blue) contained 27 genes; cluster 7 (green) contained 24 genes; and cluster 8 (violet) contained 19 genes.
b, Functional protein–protein interaction (PPI) networks constructed with the 237 genes that belonged to clusters 2 to 8. The members in each cluster are coded with the same colors as indicated above (a).
c, In the classification with k = 8, clusters 1,2,3,4,6, and 8 are shown with the number of cluster members and selective modules representing specific GO terms. Clusters 5 and 7 are shown in Fig. 4d,e.
Extended Data Fig. 6
Extended Data Fig. 6 RS5 and low-dose CORT treatment upregulates the reduced expression of NMDAR subunits in the PL of CRST mice.
a, Transcript levels of the NMDA receptor subunits NR1, NR2A, and NR2B in the PL of CON, CRST, CRST + RS5, and CRST + CORT (0.1 mg/kg) groups. Sample groups were prepared from the experiments depicted in Fig. 4g,h (n = 7-12 per group)
b–d, Immunofluorescence staining of NR1 (green) expression in the PL for the indicated groups (b), and quantification levels of NR1/DAPI (c) relative to DAPI intensity (d) (n = 4-6 animals per group; n = 209 – 359 per group). DAPI, blue.
e–h, Immunofluorescence staining of NR2A (red) and NR2B (green) expression in the PL for the indicated groups (e). DAPI, blue. Quantification levels of NR2A/DAPI (f) and NR2B/DAPI (g), and DAPI intensity (h) in individual cells (n = 4-6 animals per group; n = 231 – 402 per group).
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05, **, ##, p < 0.01 (One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Extended Data Fig. 7
Extended Data Fig. 7 Chemogenetic activation of PL glutamatergic neurons in CRST mice improves stress-induced depressive behaviors.
a–f, Experimental design (a). Mice injected in the PL with the AAV8-CaMKIIα-hM3D(Gq)-mCherry excitatory vector were subjected to CRST, followed by CNO (red arrow) or Veh (black arrow) injection. Injection of the AAV8 vector and resulting mCherry expression in the PL (b). CNO-stimulated c-Fos induction in the PL of mice carrying AAV8-CaMKIIα-hM3D(Gq)-mCherry (c and d) and AAV8-hSyn-hM3D(Gq)-mCherry (e and f) (Exp #1). CNO doses, 0.1, 1.0, or 3.0 mg/kg.
g–k, Behavioral performance in the SIT (g), TST (h), and FST (i) for the indicated groups on post-stress day 1 (Exp #2-1). K-Means clustering of individuals in the SIT x TST x FST matrix (j) and % composition of each group in the clusters (k) (n = 8 per group).
l–p, Behavioral performance in the SIT (l), TST (m), and FST (n) for the indicated groups after CNO wash-out on post-stress day 15 (Exp #2-2). K-Means clustering of individuals in the SIT x TST x FST matrix (o) and % composition of each group in the clusters (p) (n = 8 per group).
q, Basal serum CORT levels in the indicated groups on post-stress day 22 (n = 8 per group).
Data are mean ± SEM. *, difference compared to control; #, difference compared to CRST. *, #, p < 0.05, **, ##, p < 0.01 (One-way ANOVA followed by Newman-Keuls post-hoc test). See Supplementary Table 4 for statistical details.
Extended Data Fig. 8
Extended Data Fig. 8 PL neurons projecting to the dBNST, NAcc and BLA are visualized by labeling with AAV-CaMKIIα-GFP.
a–f, Experimental design (a). The anterograde tracer AAV-CaMKIIα-GFP was injected into the PL region (b and c), and two weeks later, subject mice were sacrificed and GFP expression in the brain was examined. Photomicrographs showing GFP signals in the dBNST (d), NAcc (e), BLA (f), and PVN (g) regions. High magnification of the area marked with a box is indicated on the right panel of each region. dBNST, dorsal BNST, CaA, central amygdala; BLA, basolateral amygdala; NAcc, NAc core; PVN, paraventricular nucleus of the hypothalamus.
h–l, Experimental design (h). The retrograde tracer, cholera toxin subunit B-488 (CTB488), was injected into the dBNST (i and j). A week later, mice were sacrificed and CTB488 expression in the brain was examined. Photomicrographs showing CTB488 expression in the PL (k) and ventral subiculum (vSub) (l). High magnification of the area marked with a box is indicated on the right side.